EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: Human embryonic stem cells are pluripotent cell lines usually derived from human blastocysts. Their potential critically depends on long-term proliferative capacity, developmental potential after prolonged culture, and karyotypic stability. Cell viability is an important parameter for assessing cell sample quality. Here, we elaborate the stored human embryonic stem cell lines’ viability in a ready to use form for a period of 9 years (from 2007 to 2015).

Materials and Methods: Spare pre implantation stage in vitro fertilized ovum-derived cell lines were cultured in suitable media. Thereafter, they were centrifuged at 1000 revolutions/min over 5 minutes, and pellets were suspended in normal saline. Next, they were tested for viability from storage at -20°C. After being allowed to thaw slowly, the cells were stained with propidium iodide and analyzed using flow cytometry. Images of cells were taken at ×40 and ×100 magnification.

Results: At ×100 magnification, cell population size ranged from 0.2 to 2 µm. The percentage of live cells was more than 95% throughout the 9 years. Cells frozen in 2015 showed cell viability of 96.8%.

Conclusions: We observed high cell viability in our cell lines for 9 years. Human embryonic stem cell lines in a ready-to-use form can be preserved for long-term purposes. Thus, they could be made available globally.

Key words : Freezing, Stability, Stem cells, Thawing

Human embryonic stem cell (hESC) lines are pluripotent cell lines, usually derived from the explanted inner cell mass of human blastocysts. These cell lines were first isolated and cultured by James Thomson and associates in 1998.¹ The hESC lines expressed cell surface in vitro fertilization markers that characterize undifferentiated nonhuman primate embryonic stem cells and hESCs, including stage-specific embryonic antigens 3 and 4, tumor rejection antigens l-60 and 1-81, and alkaline phosphatase.²

The major aim for development of hESCs was to use these cells for regenerative medicine because of their potential to proliferate and differentiate into any cell type of the human body, such as brain cells (neurons, astrocytes, glial cells, etc), blood cells (erythrocytes, lymphocytes, etc), liver cells, cardiac muscle cells, pancreatic cells, and skin cells.^3-8This potential has led to its widespread application in the fields of human developmental biology, drug discovery, drug testing, and tissue engineering and transplant medicine.^9-11

Furthermore, the differentiated derivatives of hESCs could be used for (1) identification of gene targets for new drugs, (2) testing the toxicity or teratogenicity of new compounds, and (3) transplant to replace cells destroyed by disease.¹² Despite their great potential, hESCs have not been a prolific source of cells for cell-based therapy. This is mainly due to the technical difficulties in cell culture. The maintenance and expansion of hESCs require laborious and skill-intensive procedures. In addition, the population-doubling time of hESCs is almost 3 times longer than that of the mouse embryonic stem cells.12 Also, the scientific and therapeutic potential of hESCs critically depends on their long-term proliferative capacity, their developmental potential after prolonged culture, and their karyotypic stability. The number of live cells in the total population of cells or cell viability is thus an important parameter to assess the quality of cell samples.

The hESC lines described here were derived from 2-cell stage preblastomeric origin. The cell line was originally isolated in 1999. These cells were taken from a fertilized ovum (50 nm-2.5 μm in size) discarded in a regular in vitro fertilization cycle, with full consent from the donor. The details on derivation and characterization of this cell line have been published in a separate paper.¹³ The cells are of 2 types: predominantly neuronal and nonneuronal (mesenchymal, ocular, germ, etc) progenitor cells with their undifferentiated states. The uniqueness of these cells is that they are in a ready-to-inject form and can be used in a number of otherwise incurable conditions. The safety and efficacy of the cell line have already been established.¹⁴ Assessment of cell viability is crucial for clinical therapy. In the present study, we will discuss the cell viability of the hESCs derived, maintained, and stored at our facility over a period of 9 years from 2007 to 2015.

Until now, various methods for assessing the cell viability have been available, such as trypan blue dye exclusion assay, metabolic assays like 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide), live/dead viability staining assays like acridine orange/propidium iodide (PI) and ethidium bromide/PI, and enzymatic assays like lactate dehydrogenase.1 Of these, trypan blue dye exclusion assay is a widely used assay. Previous studies have shown that the trypan blue dye exclusion method is highly prone to human errors, is time consuming, and is labor intensive and can be difficult for counting the large number of cell samples.¹⁵ In the present study, we used PI, a fluorescent intercalating agent, for assessing cell viability. Cells containing these dyes can be counted with the help of bright-field microscopy, epifluorescence microscopy, or flow cytometry.

Materials and Methods

Origin of cell lines
Cell lines were obtained from a single, spare, expendable, preimplantation-stage fertilized ovum taken during natural in vitro fertilization process after informed patient consent.

Culture of human embryonic stem cells
Cells were grown in a flask containing Roswell Park Memorial Institute and Dulbecco modified Eagle media added in the ratio of 1:35. Cells were incubated in T flasks at 37°C in a water-jacket incubator that maintained an atmosphere of 3.5% to 6% CO₂. Derivation and characterization of the cell lines have been described in our previous publication.¹³ The full protocol for derivation and culturing of this novel cell line has been previously discussed in our patent.¹⁶ The cells were cultured and maintained in a good manufacturing practice, good laboratory practice, and good tissue practice certified laboratory.

Freezing and thawing of cells
Cultured cells were obtained and centrifuged at 1000 revolutions/min, and the pellet was resuspended in normal saline. This was then frozen at -20°C in 0.25-, 1-, 2-, 5-, and 10-mL syringes.

Cells were thawed by placing the syringes in-between palms of the hands until they reached body-temperature level. Cells were then tested for viability, which is reported to be maintained more effectively at 4°C than at 37°C.17

Cell sampling and propidium iodide staining
Syringes of cells stored from years 2007 to 2015 were thawed, and cell viability was tested by PI staining. Propidium iodide is an intercalating membrane-impermeable dye. It fails to enter cells with intact membranes. Propidium iodide can stain both DNA and RNA; hence, PI/ribonuclease staining buffer (BD Biosciences, San Jose, CA, USA) was used. Cells were thawed and divided into 2 Eppendorf tubes, each containing ~1 million cells. Eppendorf tubes were centrifuged at 8000 revolutions/min for 20 minutes in a microcentrifuge to remove media and dimethyl sulfoxide (DMSO). One Eppendorf tube was used unstained to negate any autofluorescence in the sample, and the second Eppendorf tube was used to stain cells with PI. Next, 500 μL of PI/ribonuclease was added to the Eppendorf tube (as for ~ 1 million cells). Cells were incubated in the dark for 10 to 15 minutes before being acquired in a BD Biosciences C6 ACCURI flow cytometer. Unstained tubes were also diluted with normal saline.

Acquisition in flow cytometer and statistical method applied
Cells were acquired in a BD Biosciences C6 ACCURI flow cytometer using ACCURI C6 software. Cells were acquired at slow fluidics. A total of 30 000 cellular events were captured. To remove noise from the machine, a threshold was set at Fourier shell correlation and sparse subspace clustering. Single cells were gated with the help of height versus area graph. With this gating, doublets, triplets, and quadruplets, as well as debris and noise, can be removed from the cells of interest. Overall, 8 samples from each year from 2007 to 2015 were randomly chosen using the Microsoft Excel randomization method. Mean percentage of cell viability was calculated for each year and compared.

Phase-contrast images
Cells belonging to different years were imaged using the Nikon Ellipse E200 phase-contrast microscope. Images were taken at ×40 and ×100 magnification.

Phase-contrast images
The images show different cellular populations ranging in size from 0.2 to 2 μm. The cells appear as shiny fluorescent round-shaped particles. Small vesicle-like cells can be seen at the back in good numbers. When cells were viewed at ×100 magnification, the plasma membrane appears to be thick. Proper studies on the thickness of the plasma membrane of these cells were done (Figure 1).

The percentage of live cells throughout the 9 years was more than 95%. There was an increase in the cell viability from 2007 to 2011. Years 2010 and 2011 showed the highest percentage of live cells over the 9-year period. Samples from 2012 showed the lowest percent viability (95.1%), with second lowest seen in 2014. Cells frozen in 2015 and analyzed in the same year showed cell viability of 96.8% (Figure 2A and 2B).

Most standard cryopreserving protocols suggest the use of 10% to 20% DMSO along with serum in culture media as the freezing media,^18,19 but the concentration of DMSO becomes critical when the cells have to be used for clinical purposes.^{20, 21} The use of high percentage levels of DMSO can be toxic not only for stored cells but also for patients undergoing hESC transplant. Dimethyl sulfoxide has been known for its general adverse effects in human patients, including nausea, vomiting, and abdominal cramps.²² Dimethyl sulfoxide is used as a cryopreservative for cells to be stored at subzero temperatures for longer duration and is added before freezing of cells. It is able to penetrate the cells, binding with water molecules inside the cells. During freezing, it does not allow cells to dehydrate or shrink due to the efflux of water and hence is able to maintain the intracellular environment of cells during subzero temperatures. Also, it prevents the formation of ice crystals, which can lead to cell rupture. Almost 80% of stem cell transplant centers across the world are still using 10% DMSO.²³ Reducing the DMSO concentration to 2.2% was shown to be effective and less toxic to the cells and patients.^21,22 In our samples, DMSO is used at a much lower concentration of 0.2% to 2% to prevent any possible toxicity of DMSO to the patients. The nomenclature committee on cell death has concluded that a cell will be considered as “dead” only under 3 conditions: (1) the integrity of the plasma membrane of the cell has been lost and/or (2) the nucleus of the cell has undergone degradation and/or (3) the cell has been endocytosed by other healthy cells. Of these, assessing the integrity of the plasma membrane of the cell is the easiest and quickest method to analyze cell viability.²⁴ Most commercially available cell viability assays are also based on the same principle.

Cell death via necrosis or apoptosis leads to membrane blabbing or membrane disruption. When cells are stored at subzero temperatures for longer duration, they undergo cell death because of the freeze-thaw stress or due to the formation of ice crystals.¹⁷ Use of cell-impermeable nucleic acid stains for cell viability tests provides a true way to analyze the number of cells. Cells taking up the stains, that is, the cells whose plasma membrane has been disrupted, are dead cells, and the cells not taking the stain are the live cells with integrated membranes.²⁵ Propidium iodide binds to both RNA and DNA, and thus it is a prerequisite to add ribonuclease to the cells so as to remove RNA, which could create some confusion in the DNA assessment.²⁶ Propidium iodide works best with flow cytometer for cell cycle and cell viability assays.²⁷ It intercalates nonspecifically between the DNA bases with a stoichiometric ratio of 1 dye per 4 to 5 base pairs of DNA. Our study included the analyses of 170 samples of hESCs derived, maintained, and stored at our facility for the past 9 years. Each year, 8 random samples were selected for analyses. We do not mention trypan blue for the cell viability assay as our cells are small (ie, in the range of 0.2-2 μm). The trypan blue method is highly prone to errors. If the cells are left in trypan blue stain for a longer time period while counting them, some cells are capable of taking up the stain, thereby creating discrepancies in the actual data.15 Use of PI for cell viability assays is a quick and robust method, and large numbers of samples can be handled easily at one time.²⁷

Human embryonic stem cells are also known to be highly prone to abnormal karyotype after longer passages. Mitalipova and associates showed that BG01 and BG02 cell lines developed abnormal karyotype just after 40 passages.²⁸ Our cell line has been passaged for over 4000 passages, and the genomic integrity of the cells is maintained (unpublished results). This makes our hESC line well efficient to be used for clinical therapies.

Various other factors influence the viability of cell lines, including chromosomal instability,²⁹ tedious and sensitive passage methods,³⁰ and difficult culturing techniques. Chromosomal instability is the biggest issue in the viability of cell lines. The causes for chromosomal instability include (1) long-term culture, (2) in vitro oxygen tension, (3) adaptive pressure to culture conditions, and (4) mechanical/enzymatic pressure for cell detaching.³¹ A study by Lo and colleagues stated that chromosome instability resulted in the breakage of double strands near telomeres in embryonic stem cells.³² In our study, we did not observe chromosomal instability in our cell lines (unpublished observations).

Regarding the passage method, it has been hypothesized that mechanical passage of hESCs by cutting the colonies into small pieces may contribute to the perpetuation of the euploid population, reducing the appearance of aneuploid clones, which seem more common on enzymatic or chemical passage methodology.³⁰ Viability of cell lines is also influenced by the storage conditions. A study by Kashima and associates reported that there were significant differences in the viability of cells stored at -80°C compared with cells stored at -196°C.³³ The feeder cells during the culturing of hESC lines also play an important role in the viability of cells. Eiselleova and associates conducted a study to compare the effects of mouse and human feeder cells for hESCs. They found a significantly lower proportion of hESCs maintained on human feeder cell compared with mouse feeder cells. The authors concluded that the ability of a feeder layer to promote the undifferentiated growth of hESCs is attributable to its characteristic growth factor production.³⁴ However, in our study, we did not use any feeder cells for the culturing of the hESCs lines.

In our previous studies, hESC treatment had shown significant improvements in the condition of patients suffering from cerebral palsy, cortical visual impairment, Friedrich’s ataxia, emphysematous lung disease, and spinal cord injury.^35-38 The differentiation of embryonic stem cells has been explained and discussed in our previous paper.³⁹ Until now, we have used this cell line in > 1400 patients with various incurable conditions with remarkable results. No adverse events or teratoma formations were observed.

Our analyses of viability of these cells showed that 95% of the cells were alive after being thawed for many years. The cell samples from 2007, which had been stored for 8 years and were thawed during 2015, showed 96.1% cell viability. The cells that were frozen in 2015 and thawed in the same year showed 96.8% cell viability. The cell samples stored in 2012 showed a high number of dead cells, with 95.1% viable cells (Figure 3). There was an increase in cell viability from 2007 to 2011, which can be interpreted from the fact that cells from 2007 had been stored for almost 8 years, whereas cells from 2011 had been stored for 4 years and had less storage time, thus perhaps one reason for high cell viability percentages in 2010 and 2011. However, the same trend was not shown in the preceding years.

It has been almost 17 years since the discovery of first hESC line. However, even today, hESCs have not been used to their full potential for clinical therapies. Even today, scientists across the world are trying to establish hESCs without the use of any animal products. Although much has been achieved, the use of xenoproducts for culturing remains an issue. Our cell line is the first of its kind to have been established from a 2-cell stage pre implantation stage in vitro fertilized ovum undergoing epigenetic changes. The culturing of these cells requires no animal product, and it has not shown any adverse effects in any patient who has been transplanted with these cells.¹⁴ The cell line has had more than 4000 passages, and aliquots of each passage have been stored for future reference. Cell viability after thawing has been a reason of concern worldwide as no standard protocol for freezing and thawing of hESCs has been established due to the different responses of different hESCs to the same freezing conditions.

We have observed more than 95% cell viability in our hESC lines stored for a period of 9 years. Ready to use and highly viable hESC lines in an injectable form can herald a new era in the field of regenerative medicine.

Volume: 15 Issue: 3 June 2017

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